5-aminolevulinic acid salt, process for producing the same and use thereof

ABSTRACT

A 5-aminolevulinic acid salt which is useful in fields of microorganisms, fermentation, animals, medicaments, plants and the like; a process for producing the same; a medical composition comprising the same; and a plant activator composition comprising the same.

TECHNICAL FIELD

The present invention relates to a 5-aminolevulinic acid salt which isuseful in fields of microorganisms, fermentation, animals, medicaments,plants and the like; a process for producing the same; a medicalcomposition comprising the same; and a plant activator compositioncomprising the same.

BACKGROUND ART

It is known that 5-aminolevulinic acid is useful for V B₁₂ production,heme enzyme production, microbial culturing, porphyrin production andthe like in the field of microbial fermentation, for infectious diseasetreatment (Non-patent Reference 1), sterilization, Haemophilusdiagnosis, derivative materials, depilation, rheumatism therapy(Non-patent Reference 2), cancer therapy (Non-patent Reference 3),thrombus therapy (Non-patent Reference 4), diagnosis during canceroperation (Non-patent Reference 5), animal cell culture, UV cut, hememetabolism research, hair care, diagnosis of heavy metal toxication andporphyria, anemia prevention and the like in the field of animaltherapy, and for agricultural chemicals and the like in the field ofplants.

On the other hand, production method of 5-aminolevulinic acid is knownonly as its hydrochloride, and methods which use hippuric acid (PatentReference 1), succinic acid monoester chloride (Patent Reference 2),furfurylamine (Patent Reference 3), hydroxymethylfufural (PatentReference 4), oxovaleric acid methyl ester (Patent Reference 5) orsuccinic anhydride (Patent Reference 6) as the material have beenreported.

However, since the 5-aminolevulinic acid hydrochloride containshydrochloric acid, it is necessary to take into consideration corrosionof the apparatus and generation of a stimulation caused by the hydrogenchloride vaporized during the production process and compounding anddispersing process, so that it is preferable to take a countermeasurefor preventing these.

Also, in the case of oral administration of 5-aminolevulinic acidhydrochloride or its application to the skin in human, a scorchingstimulation is added to the tongue or skin. Accordingly, concern hasbeen directed toward a 5-aminolevulinic acid salt having smallerstimulation than that of 5-aminolevulinic acid hydrochloride, as the5-aminolevulinic acid to be used in the field of medicines.

In addition, since 5-aminolevulinic acid hydrochloride has a property topartially degrade at from 130 to 156° C. and completely degrade at 156°C., it has a problem of hardly able to withstand high temperature heatsterilization treatment.

A sterilization method by radiation exposure is known as a method forsolving this problem (Patent Reference 7), but this method requires aradiation exposure apparatus.

Accordingly, in order to carry out sterilization by a general andconvenient heat sterilization method, it is necessary to improve heatresistance of 5-aminolevulinic acid.

In addition, although 5-aminolevulinic acid hydrochloride is used in thefield of plants (Patent Reference 8), when used by mixing with silvernitrate or the like bactericide component generally used for plants,precipitation of silver chloride is generated in some cases through thereaction of 5-aminolevulinic acid hydrochloride with silver nitrate,which requires great care from the operational point of view because ofa possibility of disabling spraying of the agent due to clogging of thesprayer nozzle. Also, when an aqueous 5-aminolevulinic acidhydrochloride solution is directly applied to a fruit, coloring of thefruit sometimes becomes insufficient when chloride ion is present.

In addition, although an aqueous solution containing 5-aminolevulinicacid ion and nitrate ion has been suggested, 5-aminolevulinic acidnitrate has not been isolated yet (Non-patent Reference 6).

-   Patent Reference 1: JP-A-48-92328-   Patent Reference 2: JP-A-62-111954-   Patent Reference 3: JP-A-2-76841-   Patent Reference 4: JP-A-6-172281-   Patent Reference 5: JP-A-7-188133-   Patent Reference 6: JP-A-9-316041-   Patent Reference 7: JP-T-2001-514243-   Patent Reference 8: JP-A-4-338305-   Non-patent Reference 1: Peter W. et al., J. Am. Acad. Dermatol., 31,    678-680 (1994)-   Non-patent Reference 2: Kenneth T., U.S. Pat. No. 5,368,841 (1994)-   Non-patent Reference 3: Hillemanns P. et al., Int. J. Cancer, 85,    649-653 (2000)-   Non-patent Reference 4: Ichiro Yamada et al., Abstracts of Papers,    The Japanese Orthopedic Association (1988)-   Non-patent Reference 5: Kamasaki N. et al., Journal of Japan Society    for Laser Medicine, 22, 255-262 (2001)-   Non-patent Reference 6: Baxter C. S. et al., Toxicology And Applied    Pharmacology, 47, 477-482 (1979)

DISCLOSURE OF THE INVENTION

Problems that the Invention is to Solve

Thus, the present invention is to provide a novel 5-aminolevulinic acidsalt which has low stimulation or can withstand high temperature heatsterilization treatment, a production method thereof, a composition formedical treatment use comprising the same and a plant activatorcomposition comprising the same.

Means for Solving the Problems

By taking such actual circumstances into consideration, the presentinventors have conducted intensive studies and found as a result that a5-aminolevulinic acid salt which satisfies the above-describedrequirements can be obtained by eluting 5-aminolevulinic acid adsorbedon a cation exchange resin and mixing the eluate with phosphoric acid,nitric acid or sulfonic acid.

That is, the present invention relates to the following (1) to (23).

-   (1) A 5-aminolevulinic acid salt which is an aminolevulinic acid    salt wherein the salt is at least one salt selected from the group    consisting of phosphate, nitrate and sulfonate.-   (2) The 5-aminolevulinic acid salt according to the above-described    (I), which is an aminolevulinic acid phosphate represented by the    following formula (I):    HOCOCH₂CH₂COCH₂NH₂.HOP(O)(OR¹)_(n)(OH)_(2-n)  (I)

wherein R¹ represents a hydrogen atom, alkyl having from 1 to 18 carbonatoms, alkenyl having from 2 to 18 carbon atoms, aralkyl having from 7to 26 carbon atoms or phenyl; and n is an integer of from 0 to 2; andwherein when n is 2, the plural number of R¹ are the same or different.

-   (3) The 5-aminolevulinic acid salt according to the above-described    (2), wherein R¹ is a hydrogen atom, methyl, ethyl, n-butyl,    hexadecyl, 2-ethylhexyl, oleyl, benzyl or phenyl.-   (4) The 5-aminolevulinic acid salt according to the    above-described (2) or (3), which is in the form of an aqueous    solution.-   (5) The 5-aminolevulinic acid salt according to the    above-described (2) or (3), which is in the form of a solid.-   (6) The 5-aminolevulinic acid salt according to the above-described    (1), which is a 5-aminolevulinic acid nitrate.-   (7) The 5-aminolevulinic acid salt according to the above-described    (6), which is a solid.-   (8) The 5-aminolevulinic acid salt according to the above-described    (1), which is a 5-aminolevulinic acid sulfonate represented by the    following formula (II):    HOCOCH₂CH₂COCH₂NH₂.HOSO₂R²  (II)

wherein R² represents phenyl substituted with lower alkyl.

-   (9) The 5-aminolevulinic acid salt according to the above-described    (8), wherein the substituted phenyl is 4-methylphenyl,    2,4-dimethylphenyl or 2,5-dimethylphenyl.-   (10) The 5-aminolevulinic acid salt according to the    above-described (8) or (9), which is in the form of an aqueous    solution.-   (11) The 5-aminolevulinic acid salt according to the    above-described (8) or (9), which is in the form of a solid.-   (12) A process for producing the 5-aminolevulinic acid salt    according to any one of the above-described (2) to (5), which    comprises eluting 5-aminolevulinic acid adsorbed on a cation    exchange resin, and mixing the eluate with phosphoric acid.-   (13) The process according to the above-described (12), wherein the    5-aminolevulinic acid is eluted with aqueous ammonia.-   (14) A process for producing the 5-aminolevulinic acid salt    according to the above-described (6) or (7), which comprises eluting    5-aminolevulinic acid adsorbed on a cation exchange resin, and    mixing the eluate with nitric acid.-   (15) The process according to the above-described (14), wherein the    5-aminolevulinic acid is eluted with aqueous ammonia.-   (16) A process for producing the 5-aminolevulinic acid sulfonate    according to the above-described (8) or (9), which comprises eluting    5-aminolevulinic acid adsorbed on a cation exchange resin, and    mixing the eluate with sulfonic acid.-   (17) The process according to the above-described (16), wherein the    5-aminolevulinic acid is eluted with aqueous ammonia.-   (18) A composition for photodynamic treatment or photodynamic    diagnosis, which comprises the 5-aminolevulinic acid salt according    to any one of the above-described (1) to (11).-   (19) A plant activator composition which comprises the    5-aminolevulinic acid salt according to any one of the    above-described (1) to (11).-   (20) Use of the 5-aminolevulinic acid salt according to any one of    the above-described (1) to (11) for the manufacture of an agent for    photodynamic treatment or an agent for photodynamic diagnosis.-   (21) Use of the 5-aminolevulinic acid salt according to any one of    the above-described (1) to (11) as a plant activator.    Effect of the Invention

The 5-aminolevulinic acid salt of the present invention is a substancewhich is easy to handle, because it does not give off an offensive odoror a stimulative odor. Moreover, this shows a low stimulative natureupon the skin and tongue and its permeability through the skin and thelike is also excellent, so that a composition comprising this is usefulas an agent for photodynamic treatment or diagnosis. Still more, thishas a high decomposition point and a high heat resistance in comparisonwith its hydrochloride. According to the production method of thepresent invention, a 5-aminolevulinic acid salt can be producedconveniently and efficiently. In addition, since its chloride ionconcentration is low when made into an aqueous solution, damage bychlorine hardly occurs in administering it to plants.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing a relationship between concentration and pH ofaqueous 5-aminolevulinic acid salt solutions.

FIG. 2 is a schematic illustration of a dialysis cell.

FIG. 3 is a graph showing a result of pig skin permeability test ofphosphate and hydrochloride of 5-aminolevulinic acid.

FIG. 4 is a graph showing a result of onion epidermis permeability testof phosphate and hydrochloride of 5-aminolevulinic acid.

BEST MODE FOR CARRYING OUT THE INVENTION

In the above-described formula (I), the alkyl having from 1 to 18 carbonatoms represented by R¹ may be linear, branched or cyclic. The linear orbranched alkyl includes, for example, methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl,neopentyl, tert-pentyl, 2-methylbutyl, n-hexyl, isohexyl,3-methylpentyl, ethylbutyl, n-heptyl, 2-methylhexyl, n-octyl, isooctyl,tert-octyl, 2-ethylhexyl, 3-methylheptyl, n-nonyl, isononyl,1-methyloctyl, ethylheptyl, n-decyl, 1-methylnonyl, n-undecyl,1,1-dimethylnonyl, n-dodecyl, n-tridecyl, n-tetradecyl, n-pentadecyl,n-hexadecyl, n-heptadecyl, n-octadecyl and the like. The cyclic alkyl orthe alkyl containing a cyclic group includes, for example, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl,2-cyclopropylethyl, 2-cyclobutylethyl, 2-cyclopentylethyl,cyclohexylmethyl, 2-cyclohexylethyl, cycloheptylmethyl,2-cyclooctylethyl, 3-methylcyclohexyl, 4-methylcyclohexyl,4-ethylcyclohexyl, 2-methylcyclooctyl, 3-(3-methylcyclohexyl)propyl,2-(4-methylcyclohexyl)ethyl, 2-(4-ethylcyclohexyl)ethyl,2-(2-methylcyclooctyl)ethyl and the like. As the above-described alkylhaving from 1 to 18 carbon atoms, alkyl having from 1 to 16 carbon atomsis preferable, and methyl, ethyl, n-butyl, n-hexadecyl or 2-ethylhexylis particularly preferable.

The alkenyl having from 2 to 18 carbon atoms includes, for example,vinyl, allyl, isopropenyl, 2-butenyl, 2-methylallyl, 1,1-dimethylallyl,3-methyl-2-butenyl, 3-methyl-3-butenyl, 4-pentenyl, hexenyl, octenyl,nonenyl, decenyl, cyclopropenyl, cyclobutenyl, cyclopentenyl,cyclohexenyl, cycloheptenyl, cyclooctenyl, 4-methylcyclohexenyl,4-ethylcyclohexenyl, 2-cyclopentenylethyl, cyclohexenylmethyl,cycloheptenylmethyl, 2-cyclobutenylethyl, 2-cyclooctenylethyl,3-(4-methylcyclohexenyl)propyl, 5-(4-ethylcyclohexenyl)pentyl, oleyl,vaccenyl, linoleyl, linolenyl and the like, and oleyl is preferred.

The aralkyl having from 7 to 26 carbon atoms is preferably one which areconstituted by alkyl having from 1 to 6 carbon atoms and aryl havingfrom 6 to 20 carbon atoms. The alkyl having from 1 to 6 carbon atomsincludes, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl,isobutyl, tert-butyl, n-pentyl, n-hexyl, cyclopropyl, cyclobutyl,cyclohexyl and the like, and the aryl having from 6 to 20 carbon atomsincludes, for example, phenyl, naphthyl and the like. Among the aralkylshaving from 7 to 26 carbon atoms, benzyl or phenetyl is preferable, andbenzyl is particularly preferable. The aryl in the aralkyl may besubstituted with 1 to 3 substituents such as the above-described alkylhaving from 1 to 6 carbon atoms; alkoxy having from 1 to 6 carbon atomssuch as methoxy, ethoxy, n-propoxy, n-butoxy, isobutoxy and tert-butoxy;hydroxyl; amino, nitro, cyano; halogen such as fluorine, chlorine,bromine and iodine; carboxyl; and the like.

In the formula (II), the lower alkyl which substitutes the phenylrepresented by R² is alkyl having from 1 to 6 carbon atoms. The loweralkyl may be linear, branched or cyclic. The linear or branched alkylincludes, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl,isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, tert-pentyl,2-methylbutyl, n-hexyl, isohexyl, 3-methylpentyl, ethylbutyl and thelike, and methyl, ethyl or n-propyl is preferable, and methyl isparticularly preferable. The alkyl containing a cyclic chain includes,for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,2-cyclopropylethyl, 2-cyclobutylethyl and the like. Substitutingpositions and the number of lower alkyl are not particularly limited,but the number of lower alkyl is preferably from 1 to 3, more preferably1 or 2.

The phenyl substituted with lower alkyl includes, for example, phenylsubstituted with alkyl having from 1 to 6 carbon atoms, such as2-methylphenyl, 3-methylphenyl, 4-methylphenyl, 2,3-dimethylphenyl,2,4-dimethylphenyl, 2,5-dimethylphenyl, 2,6-dimethylphenyl,3,4-dimethylphenyl, 3,5-dimethylphenyl, 2,4,6-trimethylphenyl,3,4,5-trimethylphenyl, 2-ethylphenyl, tert-butylphenyl, pentylphenyl,neopentylphenyl, and hexylphenyl, and 4-methylphenyl, 2,4-dimethylphenylor 2,5-dimethylphenyl is particularly preferable.

The 5-aminolevulinic acid salt of the present invention may be a solidor a solution. The solid indicates a crystal, but may be a hydrate. Thesolution indicates a state in which the salt is dissolved or dispersedin a solvent including water, and its pH may be adjusted with a pHadjusting agent or the like. Also, the solvent including water may beused by mixing two more of them. The pH adjusting agent includes, forexample, buffers which use phosphoric acid, boric acid, phthalic acid,citric acid, succinic acid, tris, acetic acid, lactic acid, tartaricacid, phthalic acid, maleic acid and salts thereof, or Good's buffers.

An aqueous solution is preferable as the 5-aminolevulinic acid salt inthe form of solution. Concentration of the 5-aminolevulinic acid salt inthe aqueous solution is preferably from 0.01 wt ppm to 10 wt %, morepreferably from 0.1 wt ppm to 5 wt %, and most preferably from 1 wt ppmto 1 wt %. Also, pH of this aqueous solution is preferably from 3 to 7,more preferably from 3.5 to 7, and most preferably from 4 to 7. Inaddition, a salt other than the 5-aminolevulinic acid salt of thepresent invention may be contained in this aqueous solution, and in thatcase, the chloride ion concentration is preferably 50 mol % or less,more preferably 10 mol % or less, and most preferably 3 mol % or less.In this connection, the term, “does not contain chloride ion”, meansthat the chloride ion concentration is substantially 0 mol %, namely, itis preferable that this is equal to or lower than the detection limitwhen measured for example by ion chromatography (0.1 ppm).

The 5-aminolevulinic acid salt of the present invention can be producedby eluting 5-aminolevulinic acid adsorbed on a cation exchange resinwith an ion-containing aqueous solution, and mixing the eluate withphosphoric acid, nitric acid or sulfonic acid. In addition, the5-aminolevulinic acid salt can be obtained as a solid, by crystallizingit through the addition of a poor solvent to the mixed liquid. The5-aminolevulinic acid to be adsorbed on a cation exchange resin is notparticularly limited, and its purity and the like are not limited, too.That is, those which are produced in accordance with the methodsdescribed in JP-A-48-92328, JP-A-62-111954, JP-A-2-76841, JP-A-6-172281,JP-A-7-188133 and the like, and JP-A-11-42083, chemical reactionsolutions and fermentation liquids before purification thereof, articleson the market can also be used. In this connection, 5-aminolevulinicacid hydrochloride is preferably used.

The cation exchange resin may be either a strongly acidic cationexchange resin or a weakly acidic cation exchange resin. In addition, achelate resin can also be used suitably. Among them, a strongly acidiccation exchange resin is preferable. As the kind of the strongly acidiccation exchange resin, those in which sulfonate groups are linked topolystyrene system resins are preferable.

Adsorption of 5-aminolevulinic acid by the cation exchange resin can becarried out by passing an 5-aminolevulinic acid solution prepared bydissolving in an appropriate solvent through the cation exchange resin.Such a solvent is not particularly limited, so long as 5-aminolevulinicacid can be dissolved therein, and examples include water; dimethylsulfoxide; alcohols such as methanol, ethanol, propanol, isopropanol,butanol and isobutanol; amides such as N,N-dimethylformamide andN,N-dimethylacetamide; pyridines; and the like, and water, dimethylsulfoxide, methanol or ethanol is preferable, and water, methanol orethanol is particularly preferable. Also, two or more solvents may beused by mixing them. In addition, when a chemical reaction solution or afermentation liquid before purification is used, removal of the reactionsolvent or dilution with an appropriate solvent may be carried out. Inthis connection, pH of the above-described solvent and chemical reactionsolution or fermentation liquid before purification may be adjustedusing the above-described pH adjusting agent.

Although the ion-containing aqueous solution to be used in the elutionis not particularly limited, those in which phosphoric acids, nitricacid, sulfonic acids, hydroxides or carbonates of alkali metals oralkaline earth metals, ammonia, an amine, a compound containing aminogroup are dissolved in water are preferable, those in which lithiumhydroxide, sodium hydroxide, magnesium hydroxide, potassium hydroxide,calcium hydroxide, cesium hydroxide, barium hydroxide, ammoniumcarbonate, ammonium hydrogencarbonate, sodium carbonate, sodiumbicarbonate, potassium carbonate, sodium potassium carbonate, potassiumbicarbonate, ammonia, methylamine, dimethylamine, trimethylamine,ethylamine, diethylamine or triethylamine is dissolved in water is morepreferable, and those in which ammonia is dissolved in water isparticularly preferable. These aqueous solutions may be used incombination of two or more. Concentration of aqueous ammonia ispreferably from 0.01 to 10 N, more preferably from 0.1 to 3 N.

As the phosphoric acids to be mixed with the eluate of 5-aminolevulinicacid, a compound represented by formula (III)HOP(O)(OR¹)_(n)(OH)_(2-n)  (III)

wherein R¹ and n are as defined above, can be used. The phosphoric acidsinclude, for example, phosphoric acid; phosphoric acid monoesters suchas methyl phosphate, ethyl phosphate, n-butyl phosphate, 2-ethylhexylphosphate, hexadecyl phosphate, benzyl phosphate, oleyl phosphate, andphenyl phosphate; and phosphoric acid diesters such as dimethylphosphate, diethyl phosphate, di-n-butyl phosphate, di(2-ethylhexyl)phosphate, dihexadecyl phosphate, dibenzyl phosphate, dioleyl phosphate,and diphenyl phosphate, and methyl phosphate, ethyl phosphate, oleylphosphate, phenyl phosphate, dimethyl phosphate, diethyl phosphate,di-n-butyl phosphate, di(2-ethylhexyl) phosphate, dihexadecyl phosphate,dibenzyl phosphate, dioleyl phosphate or diphenyl phosphate isparticularly preferable. In addition, hypophosphorous acid orphosphorous acid can also be used suitably.

The phosphoric acids may be either hydrates or salts, and those whichare dissolved or dispersed in an appropriate solvent can be usedsuitably. The mixing amount of the phosphoric acids is preferably from 1to 5000 times molar quantity, more preferably from 1 to 500 times molarquantity, and most preferably from 1 to 50 times molar quantity, basedon the eluting amount of 5-aminolevulinic. acid deduced from the amountof the adsorbed 5-aminolevulinic acid. In this connection, the elutingamount of 5-aminolevulinic acid deduced from the amount of the adsorbed5-aminolevulinic acid varies depending on the kinds of the cationicexchange resin and eluent and the passing amount of the eluent, but isgenerally from 90 to 100% based on the amount of adsorbed5-aminolevulinic acid.

The nitric acid to be mixed with the eluate of 5-aminolevulinic acid maybe a salt, and those which are dissolved in an appropriate solvent canalso be used suitably. The mixing amount of nitric acid is the same asthe case of the above-described mixing amount of phosphoric acids.

The sulfonic acids to be mixed with the eluate of 5-aminolevulinic acidincludes, for example, p-toluenesulfonic acid,2,4-dimethylphenylsulfonic acid, 2,5-dimethylphenylsulfonic acid,3,5-dimethylphenylsulfonic acid, 2,4,6-trimethylphenylsulfonic acid andthe like, and p-toluenesulfonic acid, 2,4-dimethylphenylsulfonic acid or2,5-dimethylphenylsulfonic acid is particularly preferable. The sulfonicacids may be either hydrates or salts, and those which are dissolved ordispersed in an appropriate solvent can also be used suitably. Themixing amount of sulfonic acids is the same as the case of theabove-described mixing amount of phosphoric acids.

The solvent includes water; dimethyl sulfoxide; alcohols such asmethanol, ethanol, propanol, isopropanol, n-butanol and isobutanol;amides such as N,N-dimethylformamide and N,N-dimethylacetamide;pyridines; and the like, and water, dimethyl sulfoxide, methanol orethanol is preferable, and water, methanol or ethanol is particularlypreferable. Also, two or more solvents may be used by mixing them.

The poor solvent is not particularly limited, so long as a solid isprecipitated therein, and examples of such a solvent include alcoholssuch as methanol, ethanol, propanol, isopropanol, n-butanol andisobutanol; ethers such as diethyl ether, diisopropyl ether, dioxane,tetrahydrofuran and dimethoxyethane; esters such as methyl acetate,ethyl acetate, propyl acetate, isopropyl acetate and γ-butyrolactone;ketones such as acetone and methyl ethyl ketone; nitrites such asacetonitrile and benzonitrile; and the like, and methyl acetate, ethylacetate, γ-butyrolactone, acetone or acetonitrile is preferable, andmethyl acetate, γ-butyrolactone, acetone or acetonitrile is particularlypreferable. Also, two or more solvents may be used by mixing them.

Temperature for the elution by an ion-containing aqueous solution andthe mixing of the eluate with phosphoric acid, nitric acid or sulfonicacid is preferably from −20 to 60° C., more preferably from −10 to 30°C., under such conditions that the eluate and phosphoric acid, nitricacid or sulfonic acid do not solidify.

The 5-aminolevulinic acid salt of the present invention may be producedfrom a 5-aminolevulinic acid in which the amino group is protected witha hydrolysable protecting group, such as those in which the amino groupis protected with an acyl group or in which a protecting group capableof forming a 1,3-dioxo-1,3-dihydroisoindol-2-yl type molecular skeletonis linked to the amino group. In addition, the 5-aminolevulinic acidsalt of the present invention may also be prepared by a productionmethod other than that of the present invention, that is, a method inwhich 2-phenyl-4-(β-alkoxycarbonylpropionyl)oxazolin-5-one is hydrolyzedusing desired phosphoric acids, nitric acid or sulfonic acids or amethod in which a salt other than those with phosphoric acids, nitricacid and sulfonic acids, such as 5-aminolevulinic acid hydrochloride, isallowed to contact with desired phosphoric acids in a solvent. Compoundsof the above-described formula (III) can be used as the phosphoricacids, and those described in the foregoing can be used as the nitricacid, sulfonic acids and reaction solvents.

As is shown later in Examples, the 5-aminolevulinic acid salt does notgenerate offensive odors in comparison with 5-aminolevulinic acidhydrochloride, and particularly in the case of 5-aminolevulinic acidphosphate, it has a weak stimulation for the skin and tongue andmutagenicity is not found therein. In addition, it is excellent in itspermeability into the animal skin and plant epidermis. Accordingly,similar to the case of 5-aminolevulinic acid hydrochloride, a5-aminolevulinic acid salt, preferably 5-aminolevulinic acid phosphate,is useful as an agent for photodynamic treatment or photodynamicdiagnosis in animals including human. As the agent for photodynamictreatment or diagnosis, agents for the photodynamic treatment ordiagnosis of cancer, infectious disease, rheumatism, thrombus, pimpleand the like can be exemplified.

In using the 5-aminolevulinic acid salt as an agent for photodynamictreatment or diagnosis, it can be used under conventionally knownconditions, and more specifically, it can be used based on theprescriptions and methods disclosed in JP-T-2001-501970 (WO98/30242),JP-T-4-500770 (WO91/01727), JP-T-2005-501050 (WO2003/011265),JP-T-2004-506005 (WO2002/013788), JP-T-2001-518498 (WO99/17764) andJP-T-8-507755 (WO94/17797).

Specifically, a disease can be photodynamically treated by administeringan effective amount of the 5-aminolevulinic acid salt to an animal(including human) and carrying out light irradiation. Also, a diseasecan be photodynamically diagnosed by detecting fluorescence of theaffected part.

The composition for photodynamic treatment or photodynamic diagnosis,which contains the 5-aminolevulinic acid salt, can be made into dosageforms such as skin external preparations, injections, oral preparationsand suppositories. In making it into these dosage forms,pharmaceutically acceptable carriers can be used. As the carriers,water, binders, disintegrators, solubilizing agents, lubricants, bulkingagents, fillers and the like are used.

The dose varies depending on the age, body weight, symptom, therapeuticeffect, administration method, treating period of time and the like, butin general, this is administered within the range of from 10 mg to 10 g,more preferably from 100 mg to 1 g, per once per kg body weight peradult, once or several times a day.

In addition, when the 5-aminolevulinic acid salt is used, for example inplant applications, it may contain a generally used fertilizer componentand the like. As the fertilizer component, the substances disclosed inJP-A-4-338305 (U.S. Pat. No. 5,298,482, EP-A-0514776) can beexemplified.

The 5-aminolevulinic acid salt is also useful as a plant activator. Inusing it as a plant activator, it may be used under conventionally knownconditions, and specifically, it may be used for a plant by the methoddisclosed in JP-A-4-338305 (U.S. Pat. No. 5,298,482, EP-A-0514776).

More specifically, a foliage treating agent, a soil treating agent andthe like can be exemplified as the plant activator. In addition, thisagent may be absorbed prior to planting a plant or a cutting, or addedto water at the time of water culture.

When the 5-aminolevulinic acid salt is used as a foliage treating agent,it is preferable to contain the 5-aminolevulinic acid salt therein at aconcentration of from 1 to 1,000 ppm, particularly from 10 to 500 ppm,and to use this in an amount of from 10 to 100 liters, particularly from50 to 300 liters, per 10 are.

When the 5-aminolevulinic acid salt is used as a soil treating agent, itis preferable to use the 5-aminolevulinic acid salt in an amount of from1 to 1,000 g, particularly from 10 to 500 g, per 10 are.

When the 5-aminolevulinic acid salt is used as a foliage treating agentby applying it prior to plantation, it is preferable to contain the5-aminolevulinic acid salt at a concentration of from 1 to 1,000 ppm,particularly from 10 to 500 ppm, and to use this in an amount of from 10to 100 liters, particularly from 50 to 300 liters, per 10 are. In thisconnection, it is preferable also to use almost the same amount at thetime of water culture.

As the plant to be treated, cereals, vegetables, fruit trees, flowersand ornamental plants, trees, beans, potatoes, Welsh onions, pasture andthe like can be exemplified.

EXAMPLES

The present invention is described below in more detail based onExamples, although the present invention is not limited thereto.

Example 1

Production of 5-aminolevulinic acid phosphate

A column was charged with 180 ml of a strongly acidic ion exchange resin(AMBERLITE IR120B Na, manufactured by Japan Organo). The ion exchangeresin was used after converting it from sodium ion type to hydrogen iontype through hydrochloric acid treatment. Next, 20.00 g (119 mmol) of5-aminolevulinic acid hydrochloride was dissolved in 1000 ml of ionexchange water and passed through said column, and then 1000 ml of ionexchange water was passed through the same. Next, 1 N aqueous ammoniawas slowly passed through the same to collect 346 ml of yellow eluate.The thus collected eluate was added to 16 ml of 85% phosphoric acid(H₃PO₄ 238 mmol) and concentrated using an evaporator. To theconcentrated liquid, 400 ml of acetone was added, followed by vigorouslystirring with a stirrer and then allowed to stand at 4° C. for 16 hours.The thus precipitated solid was recovered by suction filtration andwashed with 500 ml of acetone. The thus obtained solid was dried underreduced pressure for 12 hours to obtain 23.04 g (101 mmol) of thesubstance of interest. Its physical property data are shown below.

Melting point: 108-109° C.

¹H-NMR (D₂O, 400 MHz) δ ppm: 2.67 (t, 2H, CH₂), 2.86 (t, 2H, CH₂), 4.08(s, 2H, CH₂)

¹³C-NMR (D₂O, 100 MHz) δ ppm: 30 (CH₂), 37 (CH₂), 50 (CH₂), 180 (CO),207 (COO)

Elemental analysis data: for C₅H₉NO₃.H₃PO₄

Calcd.: C, 26.21%; H, 5.28%; N, 6.11%.

Found: C, 25.6%; H, 5.2%; N, 6.1%.

PO₄ ³⁻ content by ion chromatography:

Calcd.: 41.45%.

Found: 43%.

Ion chromatography analysis conditions; separation column: IonPac AS12Amanufactured by Nippon Dionex, eluent: aqueous solution containingNa₂CO₃ and NaHCO₃ (Na₂CO₃: 0.5 mmol/l, NaHCO₃: 0.5 mmol/l), flow rate:1.5 ml/min., amount of introduced sample: 25 μl, column temperature: 35°C., detector: electric conductivity detector.

Example 2

Production of 5-aminolevulinic acid (di-n-butyl phosphate) salt

A column was charged with 180 ml of a strongly acidic ion exchange resin(AMBERLITE IR120B Na, manufactured by Japan Organo). The ion exchangeresin was used after converting it from sodium ion type to hydrogen iontype through hydrochloric acid treatment. Next, 20.00 g (119 mmol) of5-aminolevulinic acid hydrochloride was dissolved in 1000 ml of ionexchange water and passed through said column, and then 1000 ml of ionexchange water was passed through the same. Next, 1 N aqueous ammoniawas slowly passed through the same to collect 321 ml of yellow eluate.The thus collected eluate was added to 50.00 g (238 mmol) of di-n-butylphosphate and concentrated using an evaporator. To the concentratedliquid, 400 ml of acetone was added, followed by vigorously stirringwith a stirrer, and then the mixture was allowed to stand at −25° C. for16 hours. The thus precipitated solid was recovered by suctionfiltration. The thus obtained solid was dried under reduced pressure for12 hours to obtain 14.67 g (43 mmol) of the substance of interest. Itsphysical property data are shown below.

¹H-NMR (D₂O, 400 MHz) δ ppm: 0.75 (6H, CH₃), 1.23 (4H, CH₂), 1.41 (4H,CH₂), 2.46 (2H, CH₂), 2.59 (2H, CH₂), 3.66 (4H, CH₂), 3.80 (2H, CH₂)

¹³C-NMR (D₂O, 100 MHz) δ ppm: 14 (CH₃), 20 (CH₂), 29 (CH₂), 34.2 (CH₂),34.3 (CH₂), 36 (CH₂), 67 (CH₂O), 176 (COO), 204 (CO)

Example 3

Odor measurement of 5-aminolevulinic acid phosphate

Five subjects have directly smelled an aqueous solution of the5-aminolevulinic acid phosphate produced in Example 1 (a mixed liquid ofthe eluate from the column and phosphoric acid) and its solid, andevaluated their smells in accordance with the following criteria. Theresults are shown in Table 1.

Evaluation criteria:

-   0: Not smelled.-   1: Smelled but not unpleasant.-   2: Unpleasant smell.

Comparative Example 1

Smells were evaluated in the same manner as in Example 3, except that anaqueous solution of 5-aminolevulinic acid hydrochloride and its solidwere used. In this connection, the aqueous solution of 5-aminolevulinicacid hydrochloride was prepared using a solid of 5-aminolevulinic acidhydrochloride, hydrochloric acid and ion exchange water in such a mannerthat its 5-aminolevulinic acid and chloride ion concentrationsrespectively became the same molar concentrations of 5-aminolevulinicacid and phosphate ion concentrations of the aqueous solution of5-aminolevulinic acid phosphate of Example 1. The results are shown inTable 1.

TABLE 1 Example 3 Aqueous solution 0 0 0 0 0 Solid 0 0 0 0 0 ComparativeExample 1 Aqueous solution 2 2 2 2 2 Solid 1 1 1 1 1

Example 4

Smells were evaluated in the same manner as in Example 3, except that anaqueous solution prepared by dissolving 0.5 g of 5-aminolevulinic acidphosphate in 1 ml of water was used. The results are shown in Table 2.

Comparative Example 2

Smells were evaluated in the same manner as in Example 3, except that anaqueous solution prepared by dissolving 0.5 g of 5-aminolevulinic acidhydrochloride in 1 ml of water was used. The results are shown in Table2.

TABLE 2 Subject A B C D E Example 4 0 0 0 0 0 Comparative Example 2 1 01 1 0

Based on Tables 1 and 2, smells were not found in the aqueous solutionof 5-aminolevulinic acid phosphate in comparison with the aqueoussolution of 5-aminolevulinic acid hydrochloride. Since the anti-odormeasure and anti-corrosive gas measure necessary for producing anaqueous solution of 5-aminolevulinic acid hydrochloride were simplified,the handling was more convenient. In addition, the solid of5-aminolevulinic acid phosphate also generated no smells in comparisonwith the solid of 5-aminolevulinic acid hydrochloride, so that handlingssuch as weighing and dispensation were more convenient.

Example 5

Acidity Measurement of Aqueous 5-aminolevulinic acid phosphate Solution

Aqueous 5-aminolevulinic acid phosphate solutions and aqueous5-aminolevulinic acid hydrochloride solutions having a concentration offrom 1 to 1000 mM were respectively prepared, and their acidity wasmeasured at 25° C. using a pH meter. The results are shown in FIG. 1. Asis apparent from FIG. 1, in the case of the same concentration, acidityof the aqueous 5-aminolevulinic acid phosphate solution was lower thanthat of the aqueous 5-aminolevulinic acid hydrochloride solution.

Example 6

Stimulation Test of 5-aminolevulinic acid phosphate

Each of five subjects has evaluated the sense of taste of the5-aminolevulinic acid phosphate obtained in Example 1, in accordancewith the following criteria by directly putting 5 mg of its solid on thetongue. The results are shown in Table 3.

Evaluation criteria:

-   0: No stimulation is felt.-   1: There is a stimulation but weak.-   2: There is a strong stimulation.

Comparative Example 3

The sense of taste was evaluated in the same manner as in Example 6,except that 5 mg of solid of 5-aminolevulinic acid hydrochloride wasused. The results are shown in Table 3.

TABLE 3 Example 6 1 1 1 1 1 Comparative Example 3 2 2 2 2 2

As shown in Table 3, strong stimulation was not found in5-aminolevulinic acid phosphate in comparison with 5-aminolevulinic acidhydrochloride.

Example 7

Mutagenicity Test (Back Mutation Test) using Microorganisms (Bacteria)

A test was carried out in accordance with the “Standard of MutagenicityTests Using Microorganisms” (Ministry of Labor Notification No. 77,1988) (partial revision by Ministry of Labor Notification No. 67, 1997)and “Regarding the Tests Concerning Novel Chemical Substances and thelike” (dated Nov. 21, 2003: Yaku-Shoku-Hatsu No. 1121002, 2003.11.13Sei-Kyoku, No. 2, Kan-Ho-Ki-Hatsu No. 031121002). To 0.1 ml of asolution prepared by dissolving 5% (w/v) of 5-aminolevulinic acidphosphate in distilled water (Wako Pure Chemical Industries), 0.5 ml of0.1 M sodium-phosphate buffer (pH 7.4) (0.5 ml S9 mix in the case ofmetabolism activation test) was added, and 0.1 ml of each test strainsuspension (5 strains of histidine-less Salmonella typhimurium TA 100,TA 98, TA 1535 and TA 1537 and tryptophan-less Escherichia coli WP2 uvrAwere used (Japan Bioassay Research Center)) was further added thereto,followed by pre-incubation at 37° C. for 20 minutes while shaking. Aftercompletion of the culturing, 2.0 ml of top agar kept at 45° C. inadvance was added thereto and layered on a minimum glucose agar platemedium. In this case, 2 plates were arranged for each dosage. However, 3plates were arranged for a solvent control (negative control). Afterculturing at 37° C. for 48 hours, the presence or absence of growthinhibition of each test strain was observed under a stereoscopicmicroscope, and the number of appeared back mutation colonies wascounted. In the measurement, an inner area of about 80 mm in diameter ofa plate of 86 mm in diameter (84 mm in inner diameter) was measuredusing an automatic colony analyzer (CA-11: manufactured by SystemScience), and calculated by carrying out area correction and countingloss correction using a personal computer. However, since reliability ofthe automatic colony analyzer is reduced when the number of colonies is1,500 or more, 5 points in the plate were manually measured under thestereoscopic microscope to carry out area correction of the averagevalue. A dosage setting test was carried out on 7 dosages diluted at acommon ratio of 4, using a dosage of 5,000 μg/plate as the maximum whichis the maximum dosage defined by the guideline. As a result, regardlessof the presence or absence of S9 mix, increase of the number of backmutation colonies, by a factor of 2 times or more in comparison with thesolvent control, was not found in each strain. Growth inhibition of thestrains by this substance to be tested was not found. Precipitation ofthe substance to be tested was also not found. Thus, this test wascarried out by setting 5 dosages diluted at a common ratio of 2, using adosage of 5,000 μg/plate as the maximum which is the maximum dosagedefined by the guideline. As a result, regardless of the presence orabsence of metabolic activity, increase of the number of back mutationcolonies, by a factor of 2 times or more in comparison with the solventcontrol, was not found in each strain (Table 4), so that it wasconfirmed that the 5-aminolevulinic acid phosphate does not have themutation inducing ability.

TABLE 4 Presence or absence Dosage of The number of back mutation(colonies/plate) of metabolism 5-aminolevulinic acid Base pairsubstitution type Frameshift type activation system phosphate (μg/plate)TA100 TA1535 WP2uvrA TA98 TA1537 S9 Mix(−) Solvent control 92 108 7  728 26 23 24 4 6 83  (94) 12  (9) 23 (26) 18 (22) 5 (5) 313  87 10 25 9 382  (85) 10 (10) 36 (31) 7  (8) 1 (2) 625  84 13 27 5 1 95  (90) 6 (10)28 (28) 12  (9) 5 (3) 1250  84 8 28 8 4 114  (99) 11 (10) 33 (31) 3  (6)2 (3) 2500  76 6 25 15 2 82  (79) 5  (6) 28 (27) 17 (16) 2 (2) 5000 11511 25 9 4 99 (107) 3  (7) 36 (31) 7  (8) 1 (3) S9 Mix(+) Solvent control112 143 12 10 29 30 30 33 8 8 90 (115) 11 (11) 27 (29) 23 (29) 9 (8) 313146 11 31 13 2 122 (134) 10 (11) 17 (24) 11 (12) 7 (5) 625 107 13 40 9 396 (102) 6 (10) 24 (32) 9  (9) 9 (6) 1250 128 10 34 14 6 129 (129) 14(12) 29 (32) 27 (21) 6 (6) 2500 109 8 23 7 5 102 (106) 8  (8) 27 (25) 15(11) 3 (4) 5000 130 6 25 20 6 136 (133) 8  (7) 30 (28) 21 (21) 2 (4)Positive S9 Mix not Name AF-2 NaN₃ AF-2 AF-2 ICR-191 control requiredDosage (μg/plate) 0.01 0.5 0.01 0.1 1.0 Colonies/plate 564 203 155 5754068 580 (572) 216 (210)  149 (152)  560 (568)  3934 (4001)   S9 MixName AF-2 NaN₃ AF-2 AF-2 ICR-191 required Dosage (μg/plate) 1.0 2.0 10.00.5 2.0 Colonies/plate 1275 137 1230 661 257 1184 (1230)  138 (138) 1304 (1267)  668 (665)  267 (262)  Remarks) positive object substancesAF-2: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide, NaN₃: sodium azide,ICR-191:6-chloro-9-[3-(2-chloroethylamine)-propylamino]-2-methoxyacridinedihydrochloride, 2-AA: 2-aminoanthracene

Example 8 Acute Oral Toxicity Test

This test was carried out in accordance with the OECD Guideline No. 423“Acute Oral Toxicity-Acute Toxicity Grading Method” (adopted on Dec. 17,2001). Fasted female rats (Sprague-Dawley CD species) of 3 animals pergroup were treated with 5-aminolevulinic acid phosphate at a dose of 300mg per kg body weight. In addition, other fasted female rats of two ormore groups were treated at a dose of 2000 mg per kg body weight. Theywere observed after the administration continuously for 2 weeks. As aresult, death was not found in all of the rats (Table 5), there was nosign of systemic toxicity, general body weight gain was found in allrats (Table 6), and it was estimated that the acute oral 50% lethal dose(LD50) was larger than 2,500 mg per kg body weight.

TABLE 5 Dead animals per Animal hour after Dose No. administration Deadanimals per day after administration mg/kg Female 0.5 1 2 4 1 2 3 4 5 67 8 9 10 11 12 13 14 300 1-0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1-1 0 00 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1-2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 02000 2-0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2-1 0 0 0 0 0 0 0 0 0 0 0 00 0 0 0 0 0 2-2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3-0 0 0 0 0 0 0 0 00 0 0 0 0 0 0 0 0 0 3-1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3-2 0 0 0 00 0 0 0 0 0 0 0 0 0 0 0 0 0

TABLE 6 Body weight (g) per Dose Animal No. the number of days mg/kgFemale 0 7 14 300 1-0 205 242 263 1-1 214 262 287 1-2 221 250 289 20002-0 210 240 257 2-1 221 258 274 2-2 180 221 247 3-0 208 244 260 3-1 222259 275 3-2 214 252 271

Example 9 Acute Skin Stimulation Test

This test was carried out in accordance with the OECD Guideline No. 404“Acute Skin Stimulation/Corrosive Test” (adopted on Jul. 17, 1992) andEU Committee Instructions 92/69/EEC B4 Method Acute Toxicity (SkinStimulation). Using three New Zealand white rabbits (males), a solutionprepared by dissolving 0.5 g of 5-aminolevulinic acid phosphate in 0.5ml of distilled water (pH 3.1) was applied for 4 hours to a 2.5 cmsquare area of the shaved uninjured skin of each animal which was thenobserved for 1, 24, 48 and 72 hours. As a result, very slight red spotswere observed within 24 hours, but became normal when observed after 48hours (Tables 7 and 8). In addition, when a solution prepared bydissolving 0.5 g of 5-aminolevulinic acid phosphate in 0.5 ml ofdistilled water (pH 3.1) was applied for 3 minutes or 1 hour to a 2.5 cmsquare area of the shaved uninjured skin of one New Zealand white rabbit(male), and the animal was observed for 1, 24, 48 and 72 hours, skinstimulation was not observed, too (Tables 7 and 8). Based on this, sincethe P.I.I value (primary skin irritation index) was 0.5, it wasconfirmed that this salt is outside the classification of thestimulation classification by the current United Nations recommendationGHS and does not come under a stimulus substance. In this connection, asolution prepared as a control by dissolving 0.5 g of 5-aminolevulinicacid hydrochloride in 0.5 ml of distilled water was judged corrosive bythe OECD Guideline because its pH was 2.0 or less, so that the test wasnot carried out.

TABLE 7 Rabbit No. Condition of skin Observation (formed numbers)reaction period (hour) No. 33 No. 67 No. 68 Total Formation of 1 1 1 1 3erythema and dry 24 1 1 1 3 crust 48 0 0 0 0 72 0 0 0 0 Formation ofdropsy 1 0 0 0 0 and edema 24 0 0 0 0 48 0 0 0 0 72 0 0 0 0 Total formednumbers after 24 hours and 72 hours: 3 Primary irritation index: 3/6 =0.5

TABLE 8 Condition Rabbit No. (formed numbers) of skin Observation No. 33reaction period (hour) 3 minutes application 1 hour applicationFormation of 1 0 0 erythema and 24 0 0 dry crust 48 0 0 72 0 0 Formationof 1 0 0 dropsy and 24 0 0 edema 48 0 0 72 0 0

Example 10 Animal Epidermis Permeation Test

Using a dialysis cell (effective area 1.13 m², FIG. 2), 17 ml ofphysiological saline of pH 6.8 was stirred and kept at 37° C. in itsacceptor layer. Pretreated pig skin total layer (epidermis+dermis) wasput on a membrane filter and arranged on the dialysis cell. To its donorlayer, 0.5 ml of 1 mM aqueous 5-aminolevulinic acid phosphate solutionwas added. After 0.2 ml of the solution in the acceptor layer wascollected at a predetermined period of time, the layer was supplementedwith new physiological saline. Then, 0.05 ml of the collected sample orstandard liquid was mixed with 3.5 ml of a liquid A (1 liter of a mixedsolution of acetyl acetone/ethanol/water=15/10/75 (v/v/v) containing 4 gof sodium chloride) and 0.45 ml of a liquid B (a solution prepared bydiluting 85 ml of formalin to 1 liter with water), and the mixture washeating-treated for 30 minutes and then, after 30 minutes, cooled withwater. Thereafter, concentration of 5-aminolevulinic acid was measuredby HPLC (this was carried out under analyzing conditions of 1.0 ml/minflow rate and 25° C. temperature, using a fluorescence detector of 473nm in excitation wavelength and 363 nm in fluorescence wavelength, usingan aqueous methanol/2.5% acetic acid solution=40/60 (v/v) solution asthe eluting solution, and using Wakosil-II 5C18HG, 4.6 mφ×150 mm, as thecolumn), and each concentration was calculated from the peak area of thestandard liquid.

Next, the same test was carried out using onion epidermis instead of thepig skin and by changing concentration of the aqueous 5-aminolevulinicacid phosphate solution to 0.1 mM. The results are shown in FIGS. 3 and4. As can be understood from FIGS. 3 and 4, 5-aminolevulinic acidhydrochloride and 5-aminolevulinic acid phosphate showed similarpermeability in the pig skin and onion epidermis.

Comparative Example 4

Permeability was measured in the same manner as in Example 10, exceptthat 5-aminolevulinic acid hydrochloride was used instead of5-aminolevulinic acid phosphate.

It was confirmed by this that, although 5-aminolevulinic acidhydrochloride causes a stimulation when directly applied to the skin asshown in Example 9, 5-aminolevulinic acid phosphate does not cause theskin stimulation, and they have the same permeability into the skin,thus showing that 5-aminolevulinic acid phosphate is a salt more usefulthan 5-aminolevulinic acid hydrochloride in certain medical treatments(photodynamic treatment and photodynamic diagnosis) and plants.

Example 11 Test on the Generation of Silver Chloride Precipitation

In 10 ml of ion exchange water, 0.5 g of 5-aminolevulinic acid phosphateand 0.5 g of silver nitrate were dissolved, the mixture was allowed tostand still for 5 minutes, and conditions of the liquid was observed.Generation of the precipitate was not found. In this connection, 0.5 gof 5-aminolevulinic acid hydrochloride and 0.5 g of silver nitrate weredissolved in 10 ml of ion exchange water and allowed to stand still for5 minutes, and conditions of the liquid was observed. Generation of theprecipitate was found.

Example 12 Coloring Test of Apple

The 5-aminolevulinic acid phosphate obtained in Example 1 was dissolvedin ion exchange water to the predetermined concentration shown in thefollowing table. A spreader (“Approach BI” manufactured by MaruwaBiochemical) was added to the liquid to a concentration of 0.1% byweight. The pH was adjusted using phosphoric acid.

An aqueous 5-aminolevulinic acid hydrochloride solution was prepared inthe same manner, except that the above-described 5-aminolevulinic acidphosphate was changed to 5-aminolevulinic acid hydrochloride and thephosphoric acid for pH adjustment was changed to hydrochloric acid.

The thus prepared liquid was sprayed at a ratio of 2 liters per branchon three main branches where young fruits of an apple variety “Fuji”bored but not yet colored into red (September 15). About 2 monthsthereafter (November 6), the apples were harvested and their coloringdegree was examined. A color meter CR-200 manufactured by MINOLTA CAMERAwas used for the measurement of color. The results are shown in Table 9.

TABLE 9 Coloring (L, a, b, values) Plot L a b 5-Aminolevulinic acid 100ppm (pH 5.0) 42.37 27.45 14.54 phosphate 200 ppm (pH 5.4) 42.43 31.0614.63 200 ppm (pH 2.0) — — — 5-Aminolevulinic acid 100 ppm (pH 5.0)42.28 25.96 14.72 hydrochloride 200 ppm (pH 4.8) 42.34 30.92 14.41 200ppm (pH 2.0) — — — No treatment 5-Aminolevulinic acid (0) 42.03 25.1614.66 —: Large spots were found on the fruits.

In the Lab values in Table 9, L represents brightness, a represents redand b represents yellow. Accordingly, higher value of a means denserred. Coloring of red was denser in the case of 5-aminolevulinic acidphosphate than the case of 5-aminolevulinic acid hydrochloride.

Example 13 Plant Activating Effect

A total of 12 pots, in which 600 g of volcanic ash soil was packed in aporcelain pot of 12 cm in inner diameter, and 1 plant of a spiderwortCommelina communis grown to a height of 15 cm was planted in 1 pot, wereprepared and placed under a constant temperature environment of 20° C.,and foliar application treatment was carried out once a day using thefollowing application liquids. Conditions of the leaves 21 daysthereafter were observed. The results are summarized in Table 10.

TABLE 10 Concentration (ppm) 0 1 2 Prepared by dissolving 1 6 plants 5plants 1 plant  5-aminolevulinic acid 10 7 plants 3 plants 2 plantsphosphate in tap water 100 6 plants 4 plants 2 plants Prepared bydissolving 1 2 plants 7 plants 3 plants 5-aminolevulinic acid 10 5plants 5 plants 2 plants hydrochloride in tap water 100 5 plants 4plants 3 plants Prepared by dissolving 1 4 plants 3 plants 5 plantssodium phosphate in tap 10 2 plants 4 plants 6 plants water 100 3 plants2 plants 7 plants Tap water 3 plants 3 plants 6 plants Judging criteria:0: Abnormality was not found on the leaf surface 1: A region discoloredto yellow was found on the leaf surface 2: A necrotic region was foundon the leaf surface

Based on the results of Table 10, a plant activation effect similar toor larger than that of 5-aminolevulinic acid hydrochloride was found in5-aminolevulinic acid phosphate.

Example 14 Plant Growth Regulation Effect

Rice seeds (Akinishiki) were soaked in Benlate (manufactured by SumikaTakeda Engei) (200 times) aqueous solution for a whole day and night andthen incubated at 30° C. under a dark condition to effect hastening ofgermination. Seeds of even pigeon breast stage were selected, 10 seedswere inserted using a pair of tweezers into a groove on one expandedpolyethylene sheet, which was made using a cutter knife, and this sheetwas floated on a tall Petri dish filled with 150 ml of 5-aminolevulinicacid phosphate of respective concentrations shown in Table 11 andincubated at 25° C. for 24 hours under 5,000 lux continuous lightirradiation. The number of repetitions was set to 3 repetitions for eachconcentration. Examination was carried out three days thereafter, andlengths of the first leaf sheath and seminal root in each plot weremeasured to calculate their ratios to those in the untreated plot and tocalculate average values thereof The results are shown in Table 11.

TABLE 11 Concentration First leaf sheath Seminal root Compound name(ppm) length (%) length (%) 5-Aminolevulinic acid 1 102 106 phosphate 10106 108 100 101 101 5-Aminolevulinic acid 1 107 103 hydrochloride 10 10196 100 98 109 Untreated plot 100 100

The 5-aminolevulinic acid phosphate showed a plant growth accelerationeffect similar to or larger than that of 5-aminolevulinic acidhydrochloride.

Example 15 Salinity Tolerance Improving Effect

A porcelain pot of 12 cm in inner diameter having no drainage hole wasfilled with 600 g of upland soil, and 7 to 8 seeds of cotton seeds(variety; M-5 Acala) were sowed, covered with 1 cm in thickness of thesoil and allowed to grow in a green house. Thereafter, generalmanagement was carried out, and at the time of leaflet development, asalinity improving agent containing each of the compounds to be testedwith respective concentrations shown in Table 12 and 0.05% (v/v) of aspreader (Neoesterin: manufactured by Kumiai Chemical Industry) wasprepared and was applied to foliage at an application volume of 100liters per 10 are. Each of the compounds to be tested was set to theconcentration shown in Table 12. Four days thereafter, sodium chloridein an amount which corresponds to 0 to 1.5% by weight per soil weight asshown in Table 12 was dissolved in 30 ml of water and added dropwise tothe soil. By further continuing general cultivation, examination wascarried out 23 days thereafter. the examination was carried out by nakedeye observation, and the results of salt damage were evaluated based onthe following 6 steps. The results are shown in Table 12.

Evaluation steps:

-   0: Absolutely no salt damage is observed.-   1: Very weak salt damage is observed.-   2: Weak salt damage is observed.-   3: Obvious salt damage is observed.-   4: Strong salt damage is observed.-   5: The plant body withered up due to salt damage.

TABLE 12 NaCl treated amount Compounds tested per soil weight (wt %)[treating concentration (ppm)] 0 0.5 0.75 1 1.5 Comparative No treatment0 1 2 3 5 Example Example 5-Aminolevulinic acid 0 0 1 2 3 phosphate (10)5-Aminolevulinic acid 0 0 0 1 2 phosphate (30) 5-Aminolevulinic acid 0 12 3 4 phosphate (100) 5-Aminolevulinic acid 0 1 1 2 3 phosphate (300)Comparative 5-Aminolevulinic acid 0 1 1 1 2 Example hydrochloride (10)5-Aminolevulinic acid 0 1 2 3 3 hydrochloride (30) 5-Aminolevulinic acid0 1 1 2 3 hydrochloride (100) 5-Aminolevulinic acid 0 0 1 1 2hydrochloride (300)

As shown in Table 12, 5-aminolevulinic acid phosphate showed a salinitytolerance improving effect similar to or larger than that of5-aminolevulinic acid hydrochloride.

When chloride ion concentration in the aqueous 5-aminolevulinic acidphosphate solutions used in the above-described examples was measured byion chromatography under the following conditions, it was equal to orlower than the detection limit (0.1 ppm) in each sample.

The measuring conditions are as follows; A: separation column (IonPacAS12A manufactured by Japan Dionex), B: guard column (Ionpac AG12Amanufactured by Japan Dionex), C: eluting solution (an aqueous solutioncontaining Na₂CO₃: 3.0 mmol/l and NaHCO₃: 0.5 mmol/l), D: flow rate (1.5ml/min), E: suppressor (ASRS (recycle mode, current value 50 mA)), F:amount of introduced sample (25 μl), G: temperature of constanttemperature oven (35° C.) and H: detector (electric conductivitydetector).

Example 16 Production of 5-aminolevulinic acid nitrate

A column was charged with 180 ml of a strongly acidic ion exchange resin(AMBERLITE IR120B Na, manufactured by Japan Organo). The ion exchangeresin was used after converting it from sodium ion type to hydrogen iontype through a hydrochloric acid treatment. Next, 36.00 g (214 mmol) of5-aminolevulinic acid hydrochloride was dissolved in 1800 ml of ionexchange water and passed through said column, and then 1000 ml of ionexchange water was passed through the same. Next, 1 N aqueous ammoniawas slowly passed through the same to collect 594 ml of yellow eluate.The thus collected eluate was added to 33 ml of 60% nitric acid (HNO₃442 mmol) and concentrated using an evaporator. To the concentratedliquid, 400 ml of methyl acetate was added, followed by vigorouslystirring with a stirrer and then the mixture was allowed to stand at 4°C. for 16 hours. The thus precipitated solid was recovered by suctionfiltration and washed with 500 ml of methyl acetate. The thus obtainedsolid was dried under reduced pressure for 12 hours to obtain 31.09 g(160 mmol) of the substance of interest. Its physical property data areshown below.

Melting point: 114° C.

¹H-NMR (D₂O, 400 MHz) δ ppm: 2.75 (t, 2H, CH₂), 2.93 (t, 2H, CH₂), 4.17(s, 2H, CH₂)

¹³C-NMR (D₂O, 100 MHz) δ ppm: 30 (CH₂), 37 (CH₂), 50 (CH₂), 180 (CO),207 (COO)

Elemental analysis data: for C₅H₉NO₃.HNO₃

Calcd.: C, 30.93%; H, 5.19%; N, 14.43%.

Found: C, 30.1%; H, 5.2%; N, 14.7%.

NO₃ ⁻ content by ion chromatography:

Calcd.: 31.94%.

Found: 31%.

Ion chromatography analysis conditions; separation column: IonPac AS12Amanufactured by Nippon Dionex, eluent: aqueous solution containingNa₂CO₃ and NaHCO₃ (Na₂CO₃: 3.0 mmol/l, NaHCO₃: 0.5 mmol/l), flow rate:1.5 ml/min., amount of introduced sample: 25 μl, column temperature: 35°C., detector: electric conductivity detector.

Example 17 Odor Measurement of 5-aminolevulinic acid nitrate

Five subjects have directly smelled an aqueous solution of the5-aminolevulinic acid nitrate produced in Example 16 (a mixed liquid ofthe eluate from the column and nitric acid) and its solid, and evaluatedtheir smells in the same manner as in Example 3. The results are shownin Table 13.

Comparative Example 5

Smells were evaluated in the same manner as in Example 17, except thatan aqueous solution of 5-aminolevulinic acid hydrochloride and its solidwere used. In this connection, the aqueous solution of 5-aminolevulinicacid hydrochloride was prepared using a solid of 5-aminolevulinic acidhydrochloride, hydrochloric acid and ion exchange water in such a mannerthat its 5-aminolevulinic acid and chloride ion concentrationsrespectively became the same molar concentrations of 5-aminolevulinicacid and nitrate ion concentrations of the aqueous solution of5-aminolevulinic acid nitrate of Example 16. The results are shown inTable 13.

TABLE 13 Subjects A B C D E Example 17 Aqueous solution 0 0 0 0 0 Solid0 0 0 0 0 Comparative Example 1 Aqueous solution 2 2 2 2 2 Solid 1 1 1 11

Example 18

Smells were evaluated in the same manner as in Example 17, except thatan aqueous solution prepared by dissolving 0.5 g of 5-aminolevulinicacid nitrate in 1 ml of water was used. The results are shown in Table14.

Comparative Example 6

Smells were evaluated in the same manner as in Example 17, except thatan aqueous solution prepared by dissolving 0.5 g of 5-aminolevulinicacid hydrochloride in 1 ml of water was used. The results are shown inTable 14.

TABLE 14 Subjects A B C D E Example 18 0 0 0 0 0 Comparative Example 6 10 1 1 0

Based on Tables 13 and 14, smells were not found in the aqueous solutionof 5-aminolevulinic acid nitrate in comparison with the aqueous solutionof 5-aminolevulinic acid hydrochloride. Since the anti-odor measure andanti-corrosive gas measure necessary for producing an aqueous solutionof 5-aminolevulinic acid hydrochloride were not necessary, the handlingwas more convenient. In addition, the solid of 5-aminolevulinic acidnitrate also generated no smells in comparison with the solid of5-aminolevulinic acid hydrochloride, so that handlings such as weighingand dispensation were more convenient.

Example 19 Test on the Generation of Silver Chloride Precipitate

In 10 ml of ion exchange water, 0.5 g of 5-aminolevulinic acid nitrateand 0.5 g of silver nitrate were dissolved, the mixture was allowed tostand still for 5 minutes, and conditions of the liquid was observed.Generation of the precipitate was not found.

In this connection, 0.5 g of 5-aminolevulinic acid hydrochloride and 0.5g of silver nitrate were dissolved in 10 ml of ion exchange water andallowed to stand still for 5 minutes, and conditions of the liquid wasobserved. Generation of the precipitate was found.

Example 20 Plant Activating Effect

After 600 g of upland soil was packed in a porcelain pot of 12 cm ininner diameter, 12 grains of radish seeds were sowed therein, coveredwith 5 mm in depth of the soil and allowed to grow in a green house.Foliar application treatment was carried out once a day using thefollowing application liquids. Conditions of the leaves 21 daysthereafter were observed. The results are summarized in Table 15. Thejudging criteria are the same as of Example 13.

TABLE 15 Concentration (ppm) 0 1 2 Prepared by dissolving 1 5 plants 5plants 2 plants 5-aminolevulinic acid 10 6 plants 5 plants 1 plant nitrate in tap water 100 4 plants 6 plants 2 plants Prepared bydissolving 1 4 plants 6 plants 2 plants 5-aminolevulinic acid 10 4plants 4 plants 4 plants hydrochloride in tap water 100 3 plants 5plants 4 plants Prepared by dissolving 1 2 plants 6 plants 4 plantssodium nitrate in tap water 10 2 plants 4 plants 6 plants 100 2 plants 5plants 5 plants Tap water 1 plant  4 plants 7 plants

Based on Table 15, a plant activation effect similar to or larger thanthat of 5-aminolevulinic acid hydrochloride was found in5-aminolevulinic acid nitrate.

Example 21 Coloring Test of Apple

The 5-aminolevulinic acid nitrate obtained in Example 16 was dissolvedin ion exchange water to the predetermined concentration shown in Table16. A spreader (“Approach BI” manufactured by Maruwa Biochemical) wasadded to the liquid to a concentration of 0.1% by weight. The pH wasadjusted using nitric acid.

A solution was prepared in the same manner, except that theabove-described 5-aminolevulinic acid nitrate was changed to5-aminolevulinic acid hydrochloride and the nitric acid was changed tohydrochloric acid.

The thus prepared liquid was sprayed at a ratio of 2 liters per branchon three main branches where young fruits of an apple variety “Fuji”bored but not yet colored into red (September 15). About 2 monthsthereafter (November 6), the apples were harvested and their coloringdegree was examined. A color meter CR-200 manufactured by MINOLTA CAMERAwas used for the measurement of color. The results are shown in Table16.

TABLE 16 Coloring (L, a, b, values) Plot L a b 5-Aminolevulinic acid 100wt ppm (pH 5.0) 42.41 26.51 14.46 nitrate 200 wt ppm (pH 4.9) 42.4731.00 14.72 200 wt ppm (pH 2.0) — — — 5-Aminolevulinic acid 100 wt ppm(pH 5.0) 42.28 25.96 14.72 hydrochloride 200 wt ppm (pH 4.8) 42.34 30.9214.41 200 wt ppm (pH 2.0) — — — No treatment 5-Aminolevulinic acid 42.0325.16 14.66 (0 wt ppm) —: Large spots were found on the fruits.

In the Lab values in Table 16, L represents brightness, a represents redand b represents yellow. Accordingly, higher value of a means denserred. Coloring of red was denser in the case of 5-aminolevulinic acidnitrate than the case of 5-aminolevulinic acid hydrochloride.

Example 22 Culturing of Plankton

5-Aminolevulinic acid nitrate was added, to a concentration of 1 mM (194ppm), to 100 ml of a sterilized culture shown in Table 17 (components ofthe culture), and a Chlorella sp. was inoculated therein and cultured ona reciprocal shaker at 30° C. under aerobic and dark conditions tomeasure the amount of cells (OD 660).

5-Aminolevulinic acid hydrochloride was added, to a concentration of 1mM (168 ppm), to 100 ml of the sterilized culture shown in Table 17(components of the culture), and the Chlorella sp. was inoculatedtherein and cultured on a reciprocal shaker at 30° C. under aerobic anddark conditions to measure the amount of cells (OD 660).

The Chlorella sp. was inoculated into 100 ml of the sterilized cultureshown in Table 17 (components of the culture) and cultured on areciprocal shaker at 30° C. under aerobic and dark conditions to measurethe amount of cells (OD 660).

TABLE 17 Components of culture mg/l NaNO₃ 250 CaCl₂•2H₂O 25 MgSO₄•7H₂O75 K₂HPO₄ 75 KH₂PO₄ 175 NaCl 25 NaSiO₂•9H₂O 50 EDTA 50 FeSO₄•7H₂O 5H₃BO₄ 10 ZnSO₄•7H₂O 10 MnCl₂•4H₂O 1.5 (NH₄)₆Mo₇O₂₄•4H₂O 1 CuSO₄•5H₂O 1.5Co(NO₃)₃•6H₂O 0.5

TABLE 18 Results (amount of cells: OD 660 nm) Culture time (day)Additives 0 1 2 3 5-Aminolevulinic acid nitrate 1.8 6.5 12.6 15.05-Aminolevulinic acid hydrochloride 1.8 6.3 12.5 14.8 None 1.8 6.2 11.914.0

As is apparent from the results of Table 18, 5-aminolevulinic acidnitrate showed the same effect of 5-aminolevulinic acid hydrochloride.

When chloride ion concentration in the aqueous 5-aminolevulinic acidnitrate solutions used in the above-described examples was measured byion chromatography under the following conditions, it was equal to orlower than the detection limit (0.1 ppm) in each sample.

The measuring conditions are as follows: A: separation column (IonpacAS12A manufactured by Japan Dionex), B: guard column (Ionpac AG12Amanufactured by Japan Dionex), C: eluting solution (an aqueous solutionconsisting of Na₂CO₃: 3.0 mmol/l and NaHCO₃: 0.5 mmol/l), D: flow rate(1.5 ml/min), E: suppressor (ASRS (recycle mode, current value 50 mA)),F: amount of introduced sample (25 μl), G: temperature of constanttemperature oven (35° C.) and H: detector (electric conductivitydetector).

Example 23 Production of 5-aminolevulinic acid p-toluenesulfonate

A column was charged with 180 ml of a strongly acidic ion exchange resin(AMBERLITE IR120B Na, manufactured by Japan Organo). The ion exchangeresin was used after converting it from sodium ion type to hydrogen iontype through a hydrochloric acid treatment. Next, 36.00 g (215 mmol) of5-aminolevulinic acid hydrochloride was dissolved in 1800 ml of ionexchange water and passed through said column, and then 1000 ml of ionexchange water was passed through the same. Next, 1 N aqueous ammoniawas slowly passed through the same to collect 555 ml of yellow eluate.The thus collected eluate was mixed with 81.72 g (430 mmol) ofp-toluenesulfonic acid monohydrate and concentrated using an evaporator.To the concentrated liquid, 400 ml of acetone was added, followed byvigorously stirring with a stirrer and then the mixture was allowed tostand still at 4° C. for 16 hours. The thus precipitated solid wasrecovered by suction filtration and washed with 400 ml of acetone. Thethus obtained solid was dried under reduced pressure for 12 hours toobtain 47.78 g (158 mmol) of the substance of interest. Its physicalproperty data are shown below.

Melting point: 186° C.

¹H-NMR (D₂O, 400 MHz) δ ppm: 2.38 (s, 3H, CH₃), 2.67 (t, 2H, CH₂), 2.84(t, 2H, CH₂), 4.10 (s, 2H, CH₂), 7.34 (d, 2H, ring H), 7.69 (d, 2H, ringH)

¹³C-NMR (D₂O, 100 MHz) δ ppm: 23 (CH₃), 30 (CH₂), 37 (CH₂), 50 (CH₂),128 (ring C), 132 (ring C), 142 (ring C), 145 (ring C), 180 (CO), 207(COO)

Elemental analysis data: for C₅H₉NO₃.C₇H₈SO₃

Calcd.: C, 47.52%; H, 5.65%; N, 4.62%.

Found: C, 47.4%; H, 5.6%; N, 4.6%.

Example 24 Odor Measurement of 5-aminolevulinic acid p-toluenesulfonate

Five subjects have directly smelled an aqueous solution of the5-aminolevulinic acid p-toluenesulfonate produced in Example 23 (a mixedliquid of the eluate from the column and p-toluenesulfonic acid) and itssolid, and evaluated their smells in the same manner as in Example 3.The results are shown in Table 19.

Comparative Example 7

Smells were evaluated in the same manner as in Example 24, except thatan aqueous solution of 5-aminolevulinic acid hydrochloride and its solidwere used. In this connection, the aqueous solution of 5-aminolevulinicacid hydrochloride was prepared using a solid of 5-aminolevulinic acidhydrochloride, hydrochloric acid and ion exchange water in such a mannerthat its 5-aminolevulinic acid and chloride ion concentrationsrespectively became the same molar concentrations of 5-aminolevulinicacid and p-toluenesulfonate ion concentrations of the aqueous solutionof 5-aminolevulinic acid p-toluenesulfonate of Example 23. The resultsare shown in Table 19.

TABLE 19 Subjects A B C D E Example 24 Aqueous solution 0 0 0 0 0 Solid0 0 0 0 0 Comparative Example 7 Aqueous solution 2 2 2 2 2 Solid 1 1 1 11

Example 25

Smells were evaluated in the same manner as in Example 24, except thatan aqueous solution prepared by dissolving 0.5 g of 5-aminolevulinicacid p-toluenesulfonate in 1 ml of water was used. The results are shownin Table 20.

Comparative Example 8

Smells were evaluated in the same manner as in Example 24, except thatan aqueous solution prepared by dissolving 0.5 g of 5-aminolevulinicacid hydrochloride in 1 ml of water was used. The results are shown inTable 20.

TABLE 20 Subjects A B C D E Example 25 0 0 0 0 0 Comparative Example 8 10 1 1 0

Based on Tables 19 and 20, smells were not found in the aqueous solutionof 5-aminolevulinic acid p-toluenesulfonate in comparison with theaqueous solution of 5-aminolevulinic acid hydrochloride. Since theanti-odor measure and anti-corrosive gas measure necessary for producingaqueous solution of 5-aminolevulinic acid hydrochloride were notnecessary, the handling was more convenient. In addition, the solid of5-aminolevulinic acid p-toluenesulfonate also generated no smells incomparison with the solid of 5-aminolevulinic acid hydrochloride, sothat handlings such as weighing and dispensation were more convenient.

Example 26 Heat Resistance under Crystalline State

Melting points were measured using a melting point apparatus.

TABLE 21 Melting point (° C.) 5-Aminolevulinic acid p-toluenesulfonate186 5-Aminolevulinic acid hydrochloride 156

As shown in Table 21, holding of the solid state was superior in5-aminolevulinic acid p-toluenesulfonate than 5-aminolevulinic acidhydrochloride.

Example 27 Degradation Test by Sterilization

Firstly, 50 mg of 5-aminolevulinic acid p-toluenesulfonate or5-aminolevulinic acid hydrochloride was heat sterilized (121° C., 20minutes, 1.5 kgf/cm²). After confirming that there is no change inweight before and after the sterilization, degree of degradation of5-aminolevulinic acid before and after the sterilization was verified bythe method described in a reference (Clin. Chem., 36/8, 1494 (1990)).The results are shown in Table 22

TABLE 22 Degradation degree (%) 5-Aminolevulinic acid p-toluenesulfonate2.7 5-Aminolevulinic acid hydrochloride 6.6

As shown in Table 22, it was found that 5-aminolevulinic acidp-toluenesulfonate has lower degradability by high temperature heatsterilization treatment than the case of 5-aminolevulinic acidhydrochloride.

Example 28 Test on the Generation of Silver Chloride Precipitate

In 10 ml of ion exchange water, 0.5 g of 5-aminolevulinic acidp-toluenesulfonate and 0.5 g of silver nitrate were dissolved, themixture was allowed to stand still for 5 minutes, and conditions of theliquid was observed. Generation of the precipitate was not found.

In this connection, 0.5 g of 5-aminolevulinic acid hydrochloride and 0.5g of silver nitrate were dissolved in 10 ml of ion exchange water andallowed to stand still for 5 minutes, and conditions of the liquid wasobserved. Generation of the precipitate was found.

Example 29 Plant Activating Effect

After 600 g of upland soil was packed in a porcelain pot of 12 cm ininner diameter, 12 grains of radish seeds were sowed therein, coveredwith 5 mm in depth of the soil and allowed to grow in a green house.Foliar application treatment was carried out once a day using thefollowing application liquids. Conditions of the leaves 21 daysthereafter were observed. The results are summarized in Table 23. Thejudging criteria are the same as of Example 13.

TABLE 23 Concentration (ppm) 0 1 2 Prepared by dissolving 1 6 plants 3plants 3 plants 5-aminolevulinic acid 10 5 plants 5 plants 2 plantsp-toluenesulfonate in tap 100 7 plants 4 plants 1 plant water Preparedby dissolving 1 4 plants 6 plants 2 plants 5-aminolevulinic acid 10 4plants 4 plants 4 plants hydrochloride in tap water 100 3 plants 5plants 4 plants Prepared by dissolving 1 1 plant 2 plants 9 plantsp-toluenesulfonic acid in 10 1 plant 2 plants 9 plants tap water 100 0plant 3 plants 9 plants Tap water 1 plant 4 plants 7 plants

Based on Table 23, a plant activation effect similar to or larger thanthat of 5-aminolevulinic acid hydrochloride was found in5-aminolevulinic acid p-toluenesulfonate.

Example 30 Coloring Test of Apple

The 5-aminolevulinic acid p-toluenesulfonate obtained in Example 23 wasdissolved in ion exchange water to the predetermined concentration shownin Table 24. A spreader (“Approach B1” manufactured by MaruwaBiochemical) was added to the liquid to a concentration of 0.1% byweight. The pH was adjusted using p-toluenesulfonic acid.

A solution was prepared in the same manner, except that theabove-described 5-aminolevulinic acid p-toluenesulfonate was changed to5-aminolevulinic acid hydrochloride and the p-toluenesulfonic acid waschanged to hydrochloric acid.

The thus prepared liquid was sprayed at a ratio of 2 liters per branchon three main branches where young fruits of an apple variety “Fuji”bored but not yet colored into red (September 15). About 2 monthsthereafter (November 6), the apples were harvested and their coloringdegree was examined. A color meter CR-200 manufactured by MINOLTA CAMERAwas used for the measurement of color. The results are shown in Table24.

TABLE 24 Coloring (L, a, b, values) Plot L a b 5-Aminolevulinic 100 ppm(pH 5.0) 42.39 26.44 14.69 acid 200 ppm (pH 4.9) 42.36 30.93 14.34p-toluenesulfonate 200 ppm (pH 2.0) — — — 5-Aminolevulinic 100 ppm (pH5.0) 42.28 25.96 14.72 acid 200 ppm (pH 4.8) 42.34 30.92 14.41hydrochloride 200 ppm (pH 2.0) — — — No treatment 5-Aminolevulinic 42.0325.16 14.66 acid (0) —: Large spots were found on the fruits.

In the Lab values in Table 24, L represents brightness, a represents redand b represents yellow. Accordingly, higher value of a means denserred. Coloring of red was denser in the case of 5-aminolevulinic acidp-toluenesulfonate than the case of 5-aminolevulinic acid hydrochloride.

When chloride ion concentration in the aqueous 5-aminolevulinic acidp-toluenesulfonate solutions used in the above-described examples wasmeasured by ion chromatography under the following conditions, it wasequal to or lower than the detection limit (0.1 ppm) in each sample.

The measuring conditions are as follows: A: separation column (IonPacAS12A manufactured by Japan Dionex), B: guard column (IonPac AG12Amanufactured by Japan Dionex), C: eluting solution (an aqueous solutionconsisting of Na₂CO₃: 3.0 mmol/l and NaHCO₃: 0.5 mmol/l), D: flow rate(1.5 ml/min), E: suppressor (ASRS (recycle mode, current value 50 mA)),F: amount of introduced sample (25 μl), G: temperature of constanttemperature oven (35° C.) and H: detector (electric conductivitydetector).

While the invention has been described in detail and with reference tospecific embodiments thereof, it will be apparent to one of skills inthe art that various changes and modifications can be made thereinwithout departing from the spirit and scope thereof.

This application is based on Japanese patent application filed on Mar.30, 2004 (Japanese Patent Application No. 2004-099670), Japanese patentapplication filed on Mar. 30, 2004 (Japanese Patent Application No.2004-099671), Japanese patent application filed on Mar. 30, 2004(Japanese Patent Application No. 2004-099672), Japanese patentapplication filed on Nov. 30, 2004 (Japanese Patent Application No.2004-345661), Japanese patent application filed on Feb. 25, 2005(Japanese Patent Application No. 2004-051216), Japanese patentapplication filed on Feb. 25, 2005 (Japanese Patent Application No.2004-051217), and Japanese patent application filed on Feb. 25, 2005(Japanese Patent Application No. 2004-051218), the entire contents ofwhich are incorporated hereinto by reference.

INDUSTRIAL APPLICABILITY

The 5-aminolevulinic acid salt of the present invention is a substancewhich is easy to handle, because it does not give off an offensive odoror a stimulative odor. Moreover, this shows a low stimulative natureupon the skin and tongue and its permeability through the skin and thelike is also excellent, so that a composition comprising this is usefulas an agent for photodynamic treatment or diagnosis. Still more, thishas a high decomposition point and a high heat resistance in comparisonwith its hydrochloride. According to the production method of thepresent invention, a 5-aminolevulinic acid salt can be producedconveniently and efficiently. In addition, since its chloride ionconcentration is low when made into an aqueous solution, damage bychlorine hardly occurs in administering it to plants.

1. An isolated and purified 5-aminolevulinic acid phosphate saltrepresented by the following formula (I):HOCOCH₂CH₂COCH₂NH₂.HOP(O)(OR¹)_(n)(OH)_(2-n)  (I) wherein R¹ representsa hydrogen atom, an alkyl having from 1 to 18 carbon atoms, an alkenylhaving from 2 to 18 carbon atoms, an aralkyl having from 7 to 26 carbonatoms or phenyl; and n is an integer of from 0 to 2; and wherein when nis 2, the plural number of R¹ are the same or different.
 2. The isolatedand purified 5-aminolevulinic acid salt according to claim 1, wherein R¹is a hydrogen atom, methyl, ethyl, n-butyl, hexadecyl, 2-ethylhexyl,oleyl, benzyl or phenyl.
 3. The isolated and purified 5-aminolevulinicacid salt according to claim 1, which is in the form of an aqueoussolution.
 4. The isolated and purified 5-aminolevulinic acid saltaccording to claim 1, which is in the form of a solid.
 5. A process forproducing the isolated and purified 5-aminolevulinic acid phosphate saltaccording to claim 1, which comprises eluting 5-aminolevulinic acidadsorbed on a cation exchange resin, and mixing the eluate with aphosphoric acid represented by the following formula (III):HOP(O)(OR¹)_(n)(OH)_(2-n)  (III) wherein R¹ and n are as defined above.6. The process according to claim 5, wherein the 5-aminolevulinic acidis eluted with aqueous ammonia.
 7. A composition for photodynamictreatment or photodynamic diagnosis, which comprises the isolated andpurified 5-aminolevulinic acid phosphate salt according to claim
 1. 8. Aplant activator composition which comprises the isolated and purified5-aminolevulinic acid phosphate salt according to claim
 1. 9. A methodfor photodynamic treatment or photodynamic diagnosis, which comprisesadministering the isolated and purified 5-aminolevulinic acid phosphatesalt according to claim 1 to a subject.
 10. A method for activating aplant, which comprises applying the isolated and purified5-aminolevulinic acid phosphate salt according to claim 1 to a plant.